Methods for decreasing beta amyloid protein

ABSTRACT

Blood cholesterol levels are correlated with production of amyloid β protein (Aβ), and are predictors of populations at risk of developing AD. Methods for lowering blood cholesterol levels can be used to decrease production of Aβ, thereby decreasing the risk of developing AD. The same methods and compositions can also be used for treating individuals diagnosed with AD. Methods include administration of compounds which increase uptake of cholesterol by the liver, such as the administration of HMG CoA reductase inhibitors, administration of compounds which block endogenous cholesterol production, such as administration of HMG CoA reductase inhibitors, administration of compositions which prevent uptake of dietary cholesterol, and administration of combinations of any of these which are effective to lower blood cholesterol levels. Methods have also been developed to predict populations at risk, based on the role of cholesterol in production of Aβ. For example, individuals with Apo E4 and high cholesterol, defined as a blood cholesterol level of greater than 200 mg/dl, post menopausal women with high cholesterol levels--especially those who are not taking estrogen, or individuals which high blood cholesterol levels who are not obese are all at risk of developing AD if blood cholesterol levels are not decreased.

The United States government has certain rights in this invention byvirtue of National Institutes of Health grant number RO1NS33325 to BruceA. Yankner.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is the most common cause of dementia in theaged population. The accumulation of large numbers of senile plaquescontaining the 40-42 amino acid amyloid β protein (Aβ) is a classicpathological feature of AD. Both genetic and cell biological findingssuggest that the accumulation of Aβ in the brain is the likely cause ofAD (Yankner, B. A. (1996) Neuron 16, 921-932.; Selkoe, D. J. Science275, 630-631 (1997)). Strong genetic evidence in support of thepathogenic role of Aβ came from the observation that individuals whoinherit mutations in the amyloid precursor protein almost invariablydevelop AD at an early age. These mutations increase the production of along variant of the Aβ peptide that forms senile plaques in the brain(Goate et al., (1991) Nature 349, 704-706). Mutations and allelicvariations in other genes that cause AD, including the presenilins andapolipoprotein E, also result in increased production or deposition ofthe Aβ peptide. Reiman, et al. (1996) N.E.J.Med. 334, 752-758, reportedthat in middle age, early to mid 50's, individuals who are homozygousfor the Apo E4 gene have reduced glucose metabolism in the same regionsof the brain as in patients with Alzheimer's disease. These findingssuggest that the pathological changes in the brain associated with thisgene start early. Furthermore, individuals with Down's syndromeoverexpress the amyloid precursor protein, develop Aβ deposits in thebrain at an early age, and develop Alzheimer's disease at an early age.Finally, the Aβ protein has been demonstrated to be highly toxic tonerve cells. Thus, it is widely believed that drugs which decrease thelevels of Aβ in the brain would prevent Alzheimer's disease.

The known genetic causes of AD can account for only a small proportionof the total number of cases of AD. Most cases of AD are sporadic andoccur in the aged population. A major goal of research is theidentification of environmental factors that predispose to AD that wouldbe amenable to therapeutic measures.

It is therefore an object of the present invention to provide methodsfor predicting populations at risk of developing AD.

It is another object of the present invention to provide diagnostics andpharmaceuticals to decrease the production of amyloid β protein (Aβ),and thereby to prevent or reduce the liklihood of developing AD.

It is a further object of the present invention to providepharmaceutical treatments to treat AD in patients' having theneuropsychiatric or diagnostic criteria for AD.

SUMMARY OF THE INVENTION

Blood cholesterol levels are correlated with production of amyloid βprotein (Aβ), and are predictors of populations at risk of developingAD. Methods for lowering blood cholesterol levels can be used todecrease production of Aβ, thereby decreasing the risk of developing AD.The same methods and compositions can also be used for treatingindividuals diagnosed with AD. Methods include administration ofcompounds which increase uptake of cholesterol by the liver, such as theadministration of HMG CoA reductase inhibitors, administration ofcompounds which block endogenous cholesterol production, such asadministration of HMG CoA reductase inhibitors, administration ofcompositions which prevent uptake of dietary cholesterol, andadministration of combinations of any of these which are effective tolower blood cholesterol levels. Methods have also been developed topredict populations at risk, based on the role of cholesterol inproduction of Aβ. For example, individuals with Apo E4 and highcholesterol, defined as a blood cholesterol level of greater than 200mg/dl, post menopausal women with high cholesterol levels--especiallythose who are not taking estrogen, or individuals which high bloodcholesterol levels who are not obese are all at risk of developing AD ifblood cholesterol levels are not decreased. In the preferred embodiment,individuals with these risk factors are treated to lower bloodcholesterol levels prior to developing any mental impairmentattributable to AD, based on accepted neuropsychiatric and diagnosticcriteria in clinical practice. Treatment is based on adminstration ofone or more compositions effective to lower cholesterol blood levels atleast 10%, which is believed to be sufficient to decrease production ofAβ.

Diagnostic kits based on the discovery of these risk factors includereagents for measurement of cholesterol, total lipoproteins, and/or ApoE4.

The examples demonstrate the use of HMG CoA reductase inhibitors totreat Alzheimer's disease. Rats fed a high cholesterol diet showincreased levels of the Alzheimer's disease Aβ protein in the brain.Cholesterol has been shown to increase the amount of Aβ in human neuronsin culture. The HMG CoA reductase inhibitors reduce cholesterolproduction. Several different HMG CoA reductase inhibitors, includinglovastatin, simvastatin, fluvastatin, pravastatin and compactin,significantly inhibit the level of Aβ production in human neuronalcultures.

DETAILED DESCRIPTION OF THE INVENTION I. Methods for PredictingPopulations at Risk for AD

Individuals at increased risk for Aβ accumulation and Alzheimer'sdisease are those who carry a copy of the apolipoprotein E4 gene(Strittmatter et al., (1993) Proc. Natl. Acad. Sci. U.S.A. 90,1977-1981), those with trisomy 21 (Down's syndrome) (Mann and Esiri,(1989) J. Neurol. Sci. 89, 169-179)), and individuals who carry amutation in one of the genes that encode the amyloid precursor protein,presenilin-1 or presenilin-2 (reviewed in Yankner, 1996). In addition,individuals with a family history of Alzheimer's disease have beendocumented to be at increased risk of Alzheimer's disease (Farrer etal., (1989) Ann. Neurol. 25, 485-492; van Duijn et al., (1991) Int. J.Epidemiol. 20 (suppl 2), S13-S20), and could therefore benefit fromprophylactic treatment with an HMG CoA reductase inhibitor.

Methods have also been developed to predict populations at risk, basedon the role of cholesterol in production of Aβ. Several risk factors fordeveloping AD have been identified. These include:

(1) individuals with Apo E4 and high cholesterol, defined as a bloodcholesterol level of greater than 200 mg/dl,

(2) post menopausal women with high cholesterol, especially those whoare not taking estrogen,

(3) young individuals with high blood cholesterol levels who are notobese (age 48-65 yrs),

(4) individuals with high blood cholesterol levels who have a familyhistory of AD,

(5) individuals with high blood cholesterol levels who have a familyhistory of AD, and

(6) all adult individuals with Down's syndrome.

These individuals are all at risk of developing AD if blood cholesterollevels are not decreased. In the preferred embodiment, individuals withthese risk factors are treated to lower blood cholesterol levels priorto developing any mental impairment attributable to AD using acceptedneuropsychiatric and diagnostic criteria for probable Alzheimer'sdisease (McKhahn et al. (1984) Neurology 34:939-944).

Individuals can be screened using standard blood tests for cholesterol,ApoE4, and/or total lipoprotein levels, as well as by taking a medicaland family history. In addition, over the counter immunoassay tests canbe used by individuals who may be at risk, so that they can seek furthermedical advise. These immunoassay kits can be qualitative and/orquantitative for elevated cholesterol, total lipoproteins, and Apo E4.

II. Methods of Treatment to Decrease Production of Aβ.

Methods for lowering blood cholesterol levels can be used to decreaseproduction of Aβ, thereby decreasing the risk of developing AD. The samemethods can also be used to treat patients who have already beendiagnosed with AD. Methods include administration of compounds whichincrease uptake of cholesterol by the liver, such as the administrationof HMG CoA reductase inhibitors, administration of compounds which blockendogenous cholesterol production, such as administration of HMG CoAreductase inhibitors, administration of compositions which preventuptake of dietary cholesterol, and administration of combinations of anyof these which are effective to lower blood cholesterol levels.

The examples indicate that several different HMG CoA reductaseinhibitors reduce the production of Aβ. HMG CoA reductase inhibitors mayact to lower cholesterol at several different levels. For example, HMGCoA reductase inhibitors have been shown to lower blood cholesterollevels by upregulating liporprotein clearance receptors in the liver(Brown and Goldstein, (1986) Science 232, 34-47). In addition, HMG CoAreductase inhibitors will directly inhibit cholesterol synthesis inneurons. Since every HMG CoA reductase inhibitor tested reduces Aβproduction, it is anticipated that new members of this class of drugswill also inhibit Aβ production. Furthermore, since increased dietarycholesterol increases Aβ in the brain, drugs which act through othermechanisms to reduce cholesterol will also inhibit Aβ production.

Representative CoA reductase inhibitors include the statins, includinglovastatin, simvastatin, compactin, fluvastatin, atorvastatin,cerivastatin, and pravastin. These are typically administered orally.

Compounds which inhibit cholesterol biosynthetic enzymes, including2,3-oxidosqualene cyclase, squalene synthase, and 7-dehydrocholesterolreductase, can also be used.

Representative compositions which decrease uptake of dietary cholesterolinclude the bile acid binding resins (cholestryramine and colestipol)and the fibrates (clofibrate). Probucol, nicotinic acid, garlic andgarlic derivatives, and psyllium are also used to lower bloodcholesterol levels. Probucol and the fibrates increase the metabolism ofcholesterol-containing lipoproteins. The cholesterol-lowering mechanismof nicotinic acid is not understood.

Although the preferential route of administration of HMG CoA reductaseinhibitors would be oral, the drugs could also by administered byintravenous, subcutaneous or intramuscular routes. In some cases, directadministration into the cerebrospinal fluid may be efficacious.

III. EXAMPLES

Prior to the studies described in the following examples, therelationship between cholesterol and Aβ levels in the brain was unknown.In one study, rabbits which were fed a high cholesterol diet showedincreased immunocytochemical staining of brain neurons with an antibodyto Aβ. However, this antibody was not specific for Aβ, and couldcross-react with other metabolites of the amyloid precursor protein(Sparks, D. L. (1996) Neurobiology of Aging. 17, 291-299). The studiesin the following examples demonstrate that: rats fed a high cholesteroldiet show increased levels of the Alzheimer's disease Aβ protein in thebrain; cholesterol increases the amount of Aβ in human neurons inculture; HMG CoA reductase inhibitors reduce cholesterol production; andseveral different HMG CoA reductase inhibitors, including lovastatin,simvastatin, fluvastatin, pravastatin and compactin, significantlyinhibit the level of Aβ production in human neuronal cultures.

Example 1

Cholesterol Increases the Level of Aβ in Human Neuronal Cultures.

Busciglio et al., (1993) Proc. Nat. Acad. Sci. 90, 2092-2096, describedthe production of Aβ by human cortical neurons in culture. To determinewhether cholesterol can affect the production of Aβ, primary human braincultures were established from the cortex of 16-20 week fetal abortuses,and the neurons incubated in the absence or presence of very low densitylipoprotein (VLDL), low density lipoprotein (LDL) or high densitylipoprotein (HDL) particles isolated from human plasma. Theselipoprotein particles are the physiological vehicles for the transportof cholesterol to cells. The effects of the different lipoproteinparticles on the levels of Aβ in the human cortical cultures wasdetermined. The human cortical cultures were maintained in serum-freeDulbecco's Modified Eagle's Medium (DMEM) with N2 supplements (aserum-free supplement that supports neuronal viability). The medium wasthen changed to the same medium (controls) or medium supplemented withVLDL, LDL, or HDL particles. After incubation for 48-72 hours, Aβ wasmeasured by immunoprecipitation of the culture medium with a polyclonalantibody to Aβ (B12), followed by Western blotting with a monoclonalantibody to Aβ (6E10). The Western blots were developed either by theenhanced chemiluminescence method or by addition of an ¹²⁵ I-labeledsecondary antibody and phosphorimager scanning. The bands correspondingto the 40 and 42 amino acid form of Aβ were analyzed quantitativelyusing a computer software program. Control human cortical culturesproduced basal levels of Aβ. Exposure of the human cortical cultures toVLDL, LDL or HDL particles increased the levels of both the 40 and 42amino forms of Aβ. These results suggest that the major classes ofcholesterol-containing lipoproteins all act to increase production of Aβin human neurons.

It was then determined whether lipoprotein particles containingapolipoproteins E or A1 were able to increase Aβ production. To addressthis question, synthetic lipoprotein particles containing these proteinswere created. Particles containing either apolipoprotein E or A1increased the level of Aβ in the human cortical cultures.

These results indicate that a variety of different cholesterol carryinglipoprotein particles can increase the production of Aβ in primary humanneuronal cultures.

Example 2

Dietary Cholesterol Increases Aβ Levels in the Brain.

After establishing that cholesterol-carrying lipoprotein particlesincrease Aβ in cultures of human neurons, it was determined whetherdietary cholesterol increases the level of Aβ in the brain in vivo.Increased dietary intake of cholesterol is known to increase circulatinglevels of lipoprotein particles, which in turn increases the delivery ofcholesterol to cells. These experiments were performed on 20 month oldrats. The rats were fed a low cholesterol diet (0.1% cholesterol) or ahigh cholesterol diet (5% cholesterol). After 10 weeks, the animals weresacrificed and the cortex was removed for measurement of Aβ levels. Aβwas assayed by immunoprecipitating cortical homogenates with the Aβantibody B12, followed by Western blotting with the commerciallyavailable Aβ monoclonal antibody 4G8.

Resolution of the Aβ isolated from rat cerebral cortex byelectrophoretic separation on gels showed that Aβ levels weresignificantly increased by about 50% in the group of rats fed the highcholesterol diet relative to the group of rats fed the low cholesteroldiet. These findings indicate that dietary cholesterol increases theamount of Aβ in the brain. It is noteworthy that the approximately 50%increase in Aβ in the brain induced by a high cholesterol diet issimilar to the increase in Aβ which occurs in Down's syndrome, which isknown to predispose to the development of Alzheimer's disease.

Example 3

HMG CoA Reductase Inhibitors Inhibit the Production of Aβ by HumanNeurons.

The HMG CoA reductase inhibitors have been used in humans to decreaseplasma levels of cholesterol in patients at risk for heart disease. Thediscovery that cholesterol increases the amount of Aβ in the brain ledto this investigation to determine whether the HMG CoA reductaseinhibitors may be therapeutically efficacious for Alzheimer's disease byinhibiting the production of Aβ. Human cortical neuronal cultures wereestablished from 18 weeks gestation normal fetal cortical tissue asdescribed above and maintained in a culture medium comprised of DMEMcontaining N2 supplements. After one week, the culture medium waschanged to DMEM+N2 supplements (control), or DMEM+N2 supplements+either100 μM lovastatin, 100 μM simvastatin, 100 μM compactin, 100 μMfluvastatin, or 1 mM pravastatin, after incubation for 48 hours, thecultured cells were harvested and the levels of Aβ were assayed, asdescribed above.

Aβ was isolated from the culture medium from human cortical neuronalcultures and resolved by electrophoresis in gels. These resultsdemonstrate that human neurons treated with either lovastatin,simvastatin, compactin, fluvastatin or pravastatin have significantlydecreased levels of Aβ relative to controls. These results indicate thatHMG CoA reductase inhibitors decrease the production of Aβ by humanneurons.

The finding that HMG CoA reductase inhibitors inhibit Aβ production byhuman cortical cells supports the use of this class of drugs forreducing the levels of Aβ in individuals with Alzheimer's disease or atrisk of developing Alzheimer's disease.

Modifications and variations of the methods and compositions forprediction of the liklihood of developing AD, and for preventing and/ortreating AD, will be obvious to those skilled in the art. Thesemodifications and variations are intended to come within the scope ofthe appended claims.

We claim:
 1. A method for decreasing the production of Aβ comprisingadministering a composition which decreases blood cholesterol levels toa person with elevated cholesterol levels who is at risk of, or exhibitsthe symptoms of, Alzheimer's disease.
 2. The method of claim 1 whereinthe composition is an HMG CoA reductase inhibitor.
 3. The method ofclaim 2 wherein the composition is selected from the group consisting oflovastatin, simvastatin, fluvastatin, pravastatin, atorvastatin,cerivastatin, and compactin.
 4. The method of claim 1 wherein thecomposition inhibits uptake of dietary cholesterol.
 5. The method ofclaim 1 wherein the composition blocks or decreases endogenouscholesterol production.
 6. The method of claim 1 wherein compositionincreases cholesterol metabolism or clearance.
 7. The method of claim 1wherein the person carries the apolipoprotein E4 gene.
 8. The method ofclaim 1 wherein the person has trisomy 21 (Down's syndrome).
 9. Themethod of claim 1 wherein the person carries one or more mutations inthe genes that encode amyloid β protein, amyloid precursor protein,presenilin-1 or presenilin-2.
 10. The method of claim 1 wherein theperson has a family history of Alzheimer's disease or dementing illness.11. The method of claim 1 wherein the person is a post menopausal womanwith high cholesterol.
 12. The method of claim 1 wherein the person hashigh blood cholesterol levels who is not obese.
 13. The method of claim1 wherein the person is between 48-65 years of age.